Sustained increase of paediatric invasive Streptococcus pyogenes infections dominated by M1UK and diverse emm12 isolates, Portugal, September 2022 to May 2023

Since autumn 2022, observed numbers of paediatric invasive group A Streptococcus infections in Portugal (n = 89) were higher than in pre-COVID-19 seasons. Between September 2022 and May 2023, the dominant diagnoses were pneumonia (25/79), mostly with empyema (20/25), and sepsis (22/79). A number of cases required admission to intensive care (27/79) and surgery (35/79), and the case fatality rate was 5.1% (4/79). Genomic sequencing (n = 55) revealed multiple genetic lineages, dominated by the M1UK sublineage (26/55) and more diverse emm12 isolates (12/55).


Bacterial strains and data sets
A set of 55 bacterial isolates of paediatric invasive group A streptococcus (GAS) collected in Portugal between September 1st 2022 and May 31st 2023 were available for high-throughput sequencing (HTS).These isolates represent eight emm types (Supplementary Table S1), with the majority being emm1 or emm12.To contextualize the emm1 (n=30) and emm12 (n=12) isolates sequenced for this study, the raw sequencing reads for 135 emm1 non-invasive isolates recovered in the United Kingdom [1], 99 emm1 and 239 emm12 invasive and non-invasive isolates from Denmark [2] and 64 emm12 isolates from a diverse collection were downloaded [3] from the European Nucleotide Archive (ENA).Additionally, 5 emm12 genome assemblies and the complete genome of M1 reference strain MGAS5005 were downloaded from the NCBI RefSeq database to include in the data sets.

High-throughput sequencing
Genomic DNA was extracted from cultures of GAS grown overnight in Todd-Hewitt broth (Oxoid, Basingstoke, UK) using the PureLink genomic DNA minikit (Invitrogen, Carlsbad, CA, USA).The initial bacterial lysis step was carried out in the presence of 45 U of mutanolysin (Sigma-Aldrich, St. Louis, MO, USA) and 86 μg of hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA).Whole-genome sequencing libraries were generated using the Nextera DNA library preparation kit (Illumina, San Diego, CA, USA).The libraries were sequenced in an Illumina NextSeq 550 or NextSeq 2000 instrument.
The parC and gyrB genes were visually inspected for changes in the quinolone resistance determining region (QRDR) relative to the most frequent allele in the quinolone susceptible isolates using Geneious Prime 2022.

cgMLST analysis
The allelic profiles of the 265 emm1 and 320 emm12 genomes were determined with chewBBACA v3.2.0 [10] using default parameters and the Streptococcus pyogenes wgMLST schema available in the Chewie-NS platform [11] (https://chewbbaca.online/species/1/schemas/1).Allelic profiles of the core loci (shared by 100% of the isolates under analysis) were used to create minimum spanning trees with the goeBURST algorithm in the online version of PHYLOViZ [12,13].

Phylogenetic analysis
The multiple sequence alignment (MSA) for the translated core loci in the 265 emm1 (1,249 loci, 395,067 amino acids) and 320 emm12 (1,128 loci, 356,221 amino acids) genomes was computed with MAFFT v7.520 [14].The approximately-maximum-likelihood phylogenetic trees were inferred based on the MSA of the translated core loci with FastTree v2.1.11[15] using default parameters.Phylogenetic trees and the associated metadata were visualized and exported using the Interactive Tree Of Life platform [16].

Figure S1 -
Figure S1 -Approximately-maximum-likelihood phylogenetic tree of 265 GAS emm1 isolates based on the MSA of 1,249 core loci.Splits colored in green indicate >0.9 Shimodaira-Hasegawa support values.The lineage of each isolate is indicated on the color strip.

Figure S2 -
FigureS2-Approximately-maximum-likelihood phylogenetic tree of 320 GAS emm12 isolates based on the MSA of 1,128 core loci.Splits colored in green indicate >0.9 Shimodaira-Hasegawa support values.The dataset and ST of each isolate is indicated on the color strips.